Analysis of the Frz signal transduction system of Myxococcus xanthus shows the importance of the conserved C-terminal region of the cytoplasmic chemoreceptor FrzCD in sensing signals

The Frz chemosensory system controls directed motility in Myxococcus xanthus by regulating cellular reversal frequency. M. xanthus requires the Frz system for vegetative swarming on rich media and for cellular aggregation during fruiting body formation on starvation media. The Frz signal transduction pathway is formed by proteins that share homology with chemotaxis proteins from enteric bacteria, which are encoded in the frzA-F putative operon and the divergently transcribed frzZ gene. FrzCD, the Frz system chemoreceptor, contains a conserved C-terminal module present in methyl-accepting chemotaxis proteins (MCPs); but, in contrast to most MCPs, FrzCD is localized in the cytoplasm and the N-terminal region of FrzCD does not contain transmembrane or sensing domains, or even a linker region. Previous work on the Frz system was limited by the unavailability of deletion strains. To understand better how the Frz system functions, we generated a series of in-frame deletions in each of the frz genes as well as regions encoding the N-terminal portion of FrzCD. Analysis of mutants containing these deletions showed that FrzCD (MCP), FrzA (CheW) and FrzE (CheA-CheY) control vegetative swarming, responses to repellents and directed movement during development, thus constituting the core components of the Frz pathway. FrzB (CheW), FrzF (CheR), FrzG (CheB) and FrzZ (CheY-CheY) are required for some but not all responses. Furthermore, deletion of approximately 25 amino acids from either end of the conserved C-terminal region of FrzCD results in a constitutive signalling state of FrzCD, which induces hyper-reversals with no net cell movement. Surprisingly, deletion of the N-terminal region of FrzCD shows only minor defects in swarming. Thus, signal input to the Frz system must be sensed by the conserved C-terminal module of FrzCD and not the usual N-terminal region. These results indicate an alternative mechanism for signal sensing with this cytoplasmic MCP.     

AglZ regulates adventurous (A-) motility in Myxococcus xanthus through its interaction with the cytoplasmic receptor, FrzCD

Myxococcus xanthus moves by gliding motility powered by type IV pili (S-motility) and distributed motor complexes (A-motility). The Frz chemosensory pathway controls reversals for both motility systems. However, it is unclear how the Frz pathway can communicate with these different systems. In this article, we show that FrzCD, the Frz pathway receptor, interacts with AglZ, a protein associated with A-motility. Affinity chromatography and cross-linking experiments showed that the FrzCD–AglZ interaction occurs between the uncharacterized N-terminal region of FrzCD and the N-terminal pseudo-receiver domain of AglZ. Fluorescence microscopy showed AglZ–mCherry and FrzCD–GFP localized in clusters that occupy different positions in cells. To study the role of the Frz system in the regulation of A-motility, we constructed aglZ frzCD double mutants and aglZ frzCD pilA triple mutants. To our surprise, these mutants, predicted to show no A-motility (A^-S⁺) or no motility at all (A^-S^-), respectively, showed restored A-motility. These results indicate that AglZ modulates a FrzCD activity that inhibits A-motility. We hypothesize that AglZ–FrzCD interactions are favoured when cells are isolated and moving by A-motility and inhibited when S-motility predominates and A-motility is reduced.     

Chemotaxis mediated by NarX-FrzCD chimeras and nonadapting repellent responses in Myxococcus xanthus

Myxococcus xanthus requires gliding motility for swarming and fruiting body formation. It uses the Frz chemosensory pathway to regulate cell reversals. FrzCD is a cytoplasmic chemoreceptor required for sensing effectors for this pathway. NarX is a transmembrane sensor for nitrate from Escherichia coli. In this study, two NarX-FrzCD chimeras were constructed to investigate M. xanthus chemotaxis: NazD(F) contains the N-terminal sensory module of NarX fused to the C-terminal signalling domain of FrzCD; NazD(R) is similar except that it contains a G51R mutation in the NarX domain known to reverse the signalling output of a NarX-Tar chimera to nitrate. We report that while nitrate had no effect on the wild type, it decreased the reversal frequency of M. xanthus expressing NazD(F) and increased that of M. xanthus expressing NazD(R). These results show that directional motility in M. xanthus can be regulated independently of cellular metabolism and physiology. Surprisingly, the NazD(R) strain failed to adapt to nitrate in temporal assays as did the wild type to known repellents. The lack of temporal adaptation to negative stimuli appears to be a general feature in M. xanthus chemotaxis. Thus, the appearance of biased movements by M. xanthus in repellent gradients is likely due to the inhibition of net translocation by repellents.     

Site-specific receptor methylation of FrzCD in Myxococcus xanthus is controlled by a tetra-trico peptide repeat (TPR) containing regulatory domain of the FrzF methyltransferase

Myxococcus xanthus is a gliding bacterium with a complex life cycle that includes swarming, predation and fruiting body formation. Directed movements in M. xanthus are regulated by the Frz chemosensory system, which controls cell reversals. The Frz pathway requires the activity of FrzCD, a cytoplasmic methyl-accepting chemotaxis protein, and FrzF, a methyltransferase (CheR) containing an additional domain with three tetra trico-peptide repeats (TPRs). To investigate the role of the TPRs in FrzCD methylation, we used full-length FrzF and FrzF lacking its TPRs (FrzF^CheR) to methylate FrzCD in vitro . FrzF methylated FrzCD on a single residue, E182, while FrzF^CheR methylated FrzCD on three residues, E168, E175 and E182, indicating that the TPRs regulate site-specific methylation. E168 and E182 were predicted consensus methylation sites, but E175 is methylated on an HE pair. To determine the roles of these sites in vivo , we substituted each methylatable glutamate with either an aspartate or an alanine residue and determined the impact of the point mutants on single cell reversals, swarming and fruiting body formation. Single, double and triple methylation site mutants revealed that each site played a unique role in M. xanthus behaviour and that the pattern of receptor methylation determined receptor activity. This work also shows that methylation can both activate and inactivate the receptor.     

Regulation of directed motility in Myxococcus xanthus

Myxococcus xanthus is a Gram-negative bacterium that exhibits a complex life cycle. During vegetative growth, cells move as large swarms. However, when starved, cells aggregate into fruiting bodies and sporulate. Both vegetative swarming and developmental aggregation require gliding motility, which involves the slow movement of cells on a solid surface in the absence of flagella. The frequency of cell reversals controls the direction of movement and is regulated by the frz genes, which encode the 'frizzy' signal-transduction proteins. These proteins contain domains which bear striking similarities to the major chemotaxis proteins of the enteric bacteria: CheA, CheY, CheW, CheR, CheB and Tar. However, significant differences exist between the Myxococcus Frz proteins and the enteric Che/MCP proteins. For example, the Frz system contains three CheY-like response-regulator domains: one is present on FrzE, which also contains a CheA-like domain, and two are present on FrzZ, which is a novel protein required for attractant, but not for repellent, responses. The identification of multiple CheY homologues in this system indicates a more complex regulatory pathway than that found in the enteric bacteria. While responses to repellent stimuli appear to follow the enteric paradigm, responses to attractants during vegetative swarming and development are more complex and may involve self-generated autoattractants. The Frz signal-transduction system regulates directed motility in M. xanthus and is essential for controlling both fruiting-body development and vegetative swarming.     

Independence and interdependence of Dif and Frz chemosensory pathways in Myxococcus xanthus chemotaxis

Dif and Frz, two Myxococcus xanthus chemosensory pathways, are required in phosphatidylethanolamine (PE) chemotaxis for excitation and adaptation respectively. DifA and FrzCD, the homologues of methyl-accepting chemoreceptors in the two pathways, were examined for methylation in the context of chemotaxis and inter-pathway interactions. Evidence indicates that DifA may not undergo methylation, but signals transmitting through DifA do modulate FrzCD methylation. Results also revealed that M. xanthus possesses Dif-dependent and Dif-independent PE-sensing mechanisms. Previous studies showed that FrzCD methylation is decreased by negative chemostimuli but increased by attractants such as PE. Results here demonstrate that the Dif-dependent sensory mechanism suppresses the increase in FrzCD methylation in attractant response and elevates FrzCD methylation upon negative stimulation. In other words, FrzCD methylation is governed by opposing forces from Dif-dependent and Dif-independent sensing mechanisms. We propose that the Dif-independent but Frz-dependent PE sensing leads to increases in FrzCD methylation and subsequent adaptation, while the Dif-dependent PE signalling suppresses or diminishes the increase in FrzCD methylation to decelerate or delay adaptation. We contend that these antagonistic interactions are crucial for effective chemotaxis in this gliding bacterium to ensure that adaptation does not occur too quickly relative to the slow speed of M. xanthus movement.     

FrzCD, a methyl-accepting taxis protein from Myxococcus xanthus, shows modulated methylation during fruiting body formation.

The frizzy (frz) genes of Myxococcus xanthus are required to control directed motility during vegetative growth and fruiting body formation. FrzCD, a protein homologous to the methyl-accepting chemotaxis proteins from enteric bacteria, is modified by methylation in response to environmental conditions. Transfer of cells from rich medium to fruiting medium initially caused rapid demethylation of FrzCD. Subsequently, the amount of FrzCD increased, but most remained unmethylated. At about the time of mound formation (9 h), most of the FrzCD was converted to methylated forms. Dispersal of developing cells (10 h) in buffer led to the demethylation of FrzCD, whereas concentration of these cells caused methylation of FrzCD. Some mutants which were unable to form fruiting bodies still modified their FrzCD during incubation under conditions of starvation on a surface.     

FrzZ, a dual CheY-like response regulator, functions as an output for the Frz chemosensory pathway of Myxococcus xanthus

Myxococcus xanthus utilizes two distinct motility systems for movement (gliding) on solid surfaces: adventurous motility (A-motility) and social motility (S-motility). Both systems are regulated by the Frz signal transduction pathway, which controls cell reversals required for directed motility and fruiting body formation. The Frz chemosensory system, unlike the Escherichia coli chemotaxis system, contains proteins with multiple response regulator domains: FrzE, a CheA-CheY hybrid protein, and FrzZ, a CheY-CheY hybrid protein. Previously, the CheY domain of FrzE was hypothesized to act as the response regulator output of the Frz system. In this study, using a genetic suppressor screen, we identified FrzZ and showed FrzZ is epistatic to FrzE, demonstrating that FrzZ is the principal output component of the pathway. We constructed M. xanthus point mutations in the phosphoaccepting aspartate residues of FrzZ and demonstrated the respective roles of these residues in group and single cell motility. We also performed in vitro assays and showed rapid phosphotransfer between the CheA domain of FrzE and each of the CheY domains of FrzZ. These experiments showed that FrzZ plays a direct role as an output of the Frz chemosensory pathway and that both CheY domains of FrzZ are functional.     

Social motility in Myxococcus xanthus requires FrzS, a protein with an extensive coiled-coil domain

Gliding motility in the developmental bacterium Myxococcus xanthus involves two genetically distinct motility systems, designated adventurous (A) and social (S). Directed motility responses, which facilitate both vegetative swarming and developmental aggregation, additionally require the 'frizzy' (Frz) signal transduction pathway. In this study, we have analysed a new gene (frzS), which is positioned upstream of the frzA-F operon. Insertion mutations in frzS caused both vegetative spreading and developmental defects, including 'frizzy' aggregates in the FB strain background. The 'frizzy' phenotype was previously considered to result only from defective directed motility responses. However, deletion of the frzS gene in an A-S+ motility background demonstrated that FrzS is a new component of the S-motility system, as the A-frzS double mutant was non-spreading (A-S-). Compared with known S-motility mutants, the frzS mutants appear similar to pilT mutants, in that both produce type IV pili, extracellular fibrils and lipopolysaccharide (LPS) O-antigen, and both agglutinate rapidly in a cohesion assay. The FrzS protein has an unusual domain composition for a bacterial protein. The N-terminal domain shows similarity to the receiver domains of the two-component response regulator proteins. The C-terminal domain is composed of up to 38 heptad repeats (a b c d e f g)38, in which residues at positions a and d are predominantly hydrophobic, whereas residues at positions e and g are predominantly charged. This periodic disposition of specific residues suggests that the domain forms a long coiled-coil structure, similar to those found in the alpha-fibrous proteins, such as myosin. Overexpression of this domain in Escherichia coli resulted in the formation of an unusual striated protein lattice that filled the cells. We speculate on the role that this novel protein could play in gliding motility.     

Sensory transduction in the gliding bacterium Myxococcus xanthus

Sensory transduction in the gliding bacterium Myxococcus xanthus is mediated by the frz genes. These genes are homologous to the chemotaxis genes of enteric bacteria and control the rate of cell reversal during gliding. Sensory transduction is hypothesized to involve the recognition of substances present in the medium at the cell surface and the subsequent stimulation of a cytoplasmic methyl-accepting protein, FrzCD. Phosphorylation of FrzE is also involved in the sensory transduction pathway. Despite the similarities between the chemotaxis proteins of enteric bacteria and M. xanthus Frz proteins, fundamental differences exist between these different bacteria in terms of the ability of cells to recognize and respond to substances in their environment. The mechanism of directional switching and the nature of the gliding motor remain obscure. It is hoped that the study of the interaction of the Frz proteins will allow greater understanding of these problems.     

A CheW homologue is required for Myxococcus xanthus fruiting body development,social gliding motility,and fibril biogenesis

In bacteria with multiple sets of chemotaxis genes, the deletion of homologous genes or even different genes in the same operon can result in disparate phenotypes. Myxococcus xanthus is a bacterium with multiple sets of chemotaxis genes and/or homologues. It was shown previously that difA and difE, encoding homologues of the methyl-accepting chemoreceptor protein (MCP) and the CheA kinase, respectively, are required for M. xanthus social gliding (S) motility and development. Both difA and difE mutants were also defective in the biogenesis of the cell surface appendages known as extracellular matrix fibrils. In this study, we investigated the roles of the CheW homologue encoded by difC, a gene at the same locus as difA and difE. We showed that difC mutations resulted in defects in M. xanthus developmental aggregation, sporulation, and S motility. We demonstrated that difC is indispensable for wild-type cellular cohesion and fibril biogenesis but not for pilus production. We further illustrated the ectopic complementation of a difC in-frame deletion by a wild-type difC. The identical phenotypes of difA, difC, and difE mutants are consistent and supportive of the hypothesis that the Dif chemotaxis homologues constitute a chemotaxis-like signal transduction pathway that regulates M. xanthus fibril biogenesis and S motility.     

Methylation of FrzCD, a methyl-accepting taxis protein of Myxococcus xanthus, is correlated with factors affecting cell behavior.

Myxococcus xanthus, a nonflagellated gliding bacterium, exhibits multicellular behavior during vegetative growth and fruiting body formation. The frizzy (frz) genes are required to control directed motility for these interactions. The frz genes encode proteins that are homologous to all of the major enteric chemotaxis proteins, with the exception of CheZ. In this study, we characterized FrzCD, a protein which is homologous to the methyl-accepting chemotaxis proteins from the enteric bacteria. FrzCD, unlike the other methyl-accepting chemotaxis proteins, was found to be localized primarily in the cytoplasmic fraction of cells. FrzCD migrates as a ladder of bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reflecting heterogeneity due to methylation or demethylation and to deamidation. FrzCD was shown to be methylated in vivo when cells were exposed to yeast extract or Casitone and demethylated when starved in buffer. We used the methylation state of FrzCD as revealed by Western blot (immunoblot) analyses to search for stimuli that are recognized by the frz signal transduction system. Common amino acids, nucleotides, vitamins, and sugars were not recognized, but certain lipids and alcohols were recognized. For example, the saturated fatty acids capric acid and lauric acid stimulated FrzCD methylation, whereas a variety of other saturated fatty acids did not. Lauryl alcohol and lipoic acid also stimulated methylation, as did phospholipids containing lauric acid. In contrast, several short-chain alcohols, such as isoamyl alcohol, and some other solvents caused demethylation. The relatively high concentrations of the chemicals required for a response may indicate that these chemicals are not the relevant signals recognized by M. xanthus in nature. Isoamyl alcohol and isopropanol also had profound effects on the behavior of wild-type cells, causing them to reverse continuously. Cells of frzB, frzF, and frzG mutants also reversed continuously in the presence of isoamyl alcohol, whereas cells of frzA, frzCD, or frzE mutants did not. On the basis of the data presented, we propose a model for the frz signal transduction pathway in M. xanthus.     

Methylation of FrzCD Defines a Discrete Step in the Developmental Program of Myxococcus xanthus

Myxococcus xanthus is a gram-negative soil bacterium which undergoes fruiting body formation during starvation. The frz signal transduction system has been found to play an important role in this process. FrzCD, a methyl-accepting taxis protein homologue, shows modulated methylation during cellular aggregation, which is thought to be part of an adaptation response to an aggregation signal. In this study, we assayed FrzCD methylation in many known and newly isolated mutants defective in fruiting body formation to determine a possible relationship between the methylation response and fruiting morphology. The results of our analysis indicated that the developmental mutants could be divided into two groups based on their ability to show normal FrzCD methylation during development. Many mutants blocked early in development, i.e., nonaggregating or abnormally aggregating mutants, showed poor FrzCD methylation. The well-characterized asg, bsg, csg, and esg mutants were found to be of this type. The defects in FrzCD methylation of these signaling mutants could be partially rescued by extracellular complementation with wild-type cells or addition of chemicals which restore their fruiting body formation. Mutants blocked in late development, i.e., translucent mounds, showed normal FrzCD methylation. Surprisingly, some mutants blocked in early development also exhibited a normal level of FrzCD methylation. The characterized mutants in this group were found to be defective in social motility. This indicates that FrzCD methylation defines a discrete step in the development of M. xanthus and that social motility mutants are not blocked in these early developmental steps.     

Developmental sensory transduction in Myxococcus xanthus involves methylation and demethylation of FrzCD.

Myxococcus xanthus is a bacterium that moves by gliding motility and exhibits multicellular development (fruiting body formation). The frizzy (frz) mutants aggregate aberrantly and therefore fail to form fruiting bodies. Individual frz cells cannot control the frequency at which they reverse direction while gliding. Previously, FrzCD was shown to exhibit significant sequence similarity to the enteric methyl-accepting chemotaxis proteins. In this report, we show that FrzCD is modified by methylation and that frzF encodes the methyltransferase. We also identify a new gene, frzG, whose predicted product is homologous to that of the cheB (methylesterase) gene from Escherichia coli. Thus, although M. xanthus is unflagellated, it appears to have a sensory transduction system which is similar in many of its components to those found in flagellated bacteria.     

Sensory adaptation during negative chemotaxis in Myxococcus xanthus.

Myxococcus xanthus exhibits many tactic movements that require the frz signal transduction system, such as colony swarming and cellular aggregation during fruiting body formation. Previously we demonstrated that the Frz proteins control the chemotactic movements of M. xanthus (W. Shi, T. Köhler, and D. R. Zusman, Mol. Microbiol. 9:601-611, 1993). However it was unclear from that study how chemotaxis might be achieved at the cellular level. In this study, we showed that M. xanthus cells not only modulate the reversal frequency of cell movement in response to repellent stimuli but also exhibit sensory adaptation in response to the continuous presence of nonsaturating repellent stimuli. The sensory adaptation behavior requires FrzF (a putative methyltransferase) and is correlated with the methylation-demethylation of FrzCD, a methyl-accepting chemotaxis protein. These results indicate that negative chemotaxis in M. xanthus is achieved by chemokinesis plus sensory adaptation in a manner analogous to that of the free-swimming enteric bacteria.     

Motility in Myxococcus xanthus and its role in developmental aggregation

The Frz signal transduction system of Myxococcus xanthus was originally thought to be a simple variation of the well-characterized Che system of the enteric bacteria. Recently, however, many additional Frz proteins, along with alternative signal transduction systems, have been discovered. Together these signal transduction pathways coordinate cell-cell behavior, permitting the complex interactions required for developmental aggregation and fruiting body formation.     

Divergent regulatory pathways control A and S motility in Myxococcus xanthus through FrzE, a CheA-CheY fusion protein

Myxococcus xanthus moves on solid surfaces by using two gliding motility systems, A motility for individual-cell movement and S motility for coordinated group movements. The frz genes encode chemotaxis homologues that control the cellular reversal frequency of both motility systems. One of the components of the core Frz signal transduction pathway, FrzE, is homologous to both CheA and CheY from the enteric bacteria and is therefore a novel CheA-CheY fusion protein. In this study, we investigated the role of this fusion protein, in particular, the CheY domain (FrzECheY). FrzECheY retains all of the highly conserved residues of the CheY superfamily of response regulators, including Asp709, analogous to phosphoaccepting Asp57 of Escherichia coli CheY. While in-frame deletion of the entire frzE gene caused both motility systems to show a hyporeversal phenotype, in-frame deletion of the FrzECheY domain resulted in divergent phenotypes for the two motility systems: hyperreversals of the A-motility system and hyporeversals of the S-motility system. To further investigate the role of FrzECheY in A and S motility, point mutations were constructed such that the putative phosphoaccepting residue, Asp709, was changed from D to A (and was therefore never subject to phosphorylation) or E (possibly mimicking constitutive phosphorylation). The D709A mutant showed hyperreversals for both motilities, while the D709E mutant showed hyperreversals for A motility and hyporeversal for S motility. These results show that the FrzECheY domain plays a critical signaling role in coordinating A and S motility. On the basis of the phenotypic analyses of the frzE mutants generated in this study, a model is proposed for the divergent signal transduction through FrzE in controlling and coordinating A and S motility in M. xanthus.     

Myxococcus xanthus swarms are driven by growth and regulated by a pacemaker

The principal social activity of Myxococcus xanthus is to organize a dynamic multicellular structure, known as a swarm. Although its cell density is high, the swarm can grow and expand rapidly. Within the swarm, the individual rod-shaped cells are constantly moving, transiently interacting with one another, and independently reversing their gliding direction. Periodic reversal is, in fact, essential for creating a swarm, and the reversal frequency controls the rate of swarm expansion. Chemotaxis toward nutrient has been thought to drive swarming, but here the nature of swarm growth and the impact of genetic deletions of members of the Frz family of proteins suggest otherwise. We find that three cytoplasmic Frz proteins, FrzCD, FrzF, and FrzE, constitute a cyclic pathway that sets the reversal frequency. Within each cell these three proteins appear to be connected in a negative-feedback loop that produces oscillations whose frequencies are finely tuned by methylation and by phosphorylation. This oscillator, in turn, drives MglAB, a small G-protein switch, to oscillate between its GTP- and GDP-bound states that ultimately determine when the cell moves forward or backward. The periodic reversal of interacting rod-shaped cells promotes their alignment. Swarm organization ensures that each cell can move without blocking the movement of others.     

The Myxococcus xanthus lipopolysaccharide O-antigen is required for social motility and multicellular development

The gliding bacterium Myxococcus xanthus aggregates to form spore-filled fruiting bodies when nutrients are limiting. Defective fruiting-body formation and sporulation result from mutations in the sasA locus, which encodes the wzm wzt wbgA (formerly rfbABC ) lipopolysaccharide (LPS) O-antigen biosynthesis genes. Mutants carrying these same sasA mutations are defective in social motility and form small glossy colonies. We report here that the developmental and motility phenotypes of four mutants each containing different Tn 5 insertions in LPS O-antigen biosynthesis genes are similar to those of the original sasA locus mutants. All of the LPS O-antigen mutants tested exhibited defective developmental aggregation and sporulated at only 0.02–15% of the wild-type level. In addition, all of the LPS O-antigen mutants were determined by genetic analyses to be wild type for adventurous motility and defective in social motility, indicating that the LPS O-antigen is necessary for normal development and social motility. The two previously identified cell-surface components required for social motility, type IV pili and the protein-associated polysaccharide material termed fibrils, were detected on the surfaces of all of the LPS O-antigen mutants. This indicates that LPS O-antigen is a third cell-surface component required for social motility.